After a unilateral muscle exercise, the performance of the non-exercised contralateral limb muscle can be also impaired. This crossover fatigue phenomenon is still debated in the literature and very few studies have investigated the influence of eccentric contractions. This study was designed to assess neuromuscular adaptations involved in the crossover fatigue of the non-exercised contralateral hamstrings. Seventeen healthy young men performed a unilateral submaximal eccentric exercise of the knee flexors until a 20% reduction in maximal voluntary isometric contraction (MVIC) torque was attained in the exercised limb (EL). Before, immediately after exercise cessation (POST) and 24 hours later, neuromuscular function, global perceived fatigue and perceived muscle soreness were measured in both the EL and non-exercised limb (NEL). At POST, significant reductions in MVIC were observed in the EL (-28.1%, p < 0.001) and in the NEL (-8.5, p < 0.05). Voluntary activation decreased (-6.0, p < 0.05) in the EL only, while potentiated doublet torque were impaired (Dt100Hz -11.6%, p < 0.001 and Dt10Hz -8.1%, p < 0.05) in both the EL and the NEL. Global perceived fatigue significantly increased at POST (p < 0.001). Interestingly, peripheral alterations and global perceived fatigue may account for the crossover fatigue observed immediately after the exercise in the NEL possibly involving systemic adaptations.